Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice Emily V. Howman, Kerry J. Fowler, Ainsley J. Newson, Saara Redward, Andrew C. MacDonald, Paul Kalitsis, and K. H. Andy Choo* The Murdoch Institute, Royal Children’s Hospital, Flemington Road, Parkville 3052, Australia Edited by John A. Carbon, University of California, Santa Barbara, CA, and approved November 23, 1999 (received for review October 5, 1999) Centromere protein A (Cenpa for mouse, CENP-A for other species) reveals embryonic lethality at 2.5 days pc, accompanied by is a histone H3-like protein that is thought to be involved in the enlarged nuclei containing an increased number of nucleoli, nucleosomal packaging of centromeric DNA. Using gene targeting, nuclear bridging, chromosome condensation, and spindle fiber we have disrupted the mouse Cenpa gene and demonstrated that bundling (21). the gene is essential. Heterozygous mice are healthy and fertile The role of CENP-A in centromeric function has yet to be whereas null mutants fail to survive beyond 6.5 days postconcep- elucidated. CENP-A is a histone-H3 like protein that is con- tion. Affected embryos show severe mitotic problems, including served in mammals (22, 23) and S. cerevisiae (24). The C terminal micronuclei and macronuclei formation, nuclear bridging and bleb- (residues 48–135) of CENP-A is 62% identical to that of histone bing, and chromatin fragmentation and hypercondensation. Im- H3 and corresponds to the histone fold domain. The histone fold munofluorescence analysis of interphase cells at day 5.5 reveals domain consists of three ␣-helices (HI, HII, and HIII) separated complete Cenpa depletion, diffuse Cenpb foci, absence of discrete by two ␤-sheet structures (strand A and strand B) (25) (see Fig. Cenpc signal on centromeres, and dispersion of Cenpb and Cenpc 1). This domain of histone H3 has been shown to be sufficient throughout the nucleus. These results suggest that Cenpa is es- for nucleosome assembly in vitro (26) and in vivo (27). There is sential for kinetochore targeting of Cenpc and plays an early role no similarity seen between the N-termini sequences (residues in organizing centromeric chromatin at interphase. The evidence is 1–47) of CENP-A and normal histone H3 (2). Although this consistent with the proposal of a critical epigenetic function for divergence initially was thought to provide CENP-A with the CENP-A in marking a chromosomal region for centromere forma- centromere targeting property, a histone H3 chimeric protein tion. containing the N terminus of CENP-A and the histone H3 histone fold domain failed to localize to the centromere, indi- kinetochore ͉ epigenetic ͉ gene targeting cating that the C-terminal end is responsible for centromere targeting (28). CENP-A synthesis appears to be coupled with he centromere is an essential chromosomal component centromere replication during mid-S to early G2 phase, whereas Trequired for the faithful segregation of chromosomes during histone H3 expression peaks early in S phase (28). Expression of mitosis and meiosis. The kinetochore is a DNA-protein complex CENP-A under the histone H3 promoter fails to localize at the comprising both constitutive proteins that are present at the centromere (28). These studies suggest that CENP-A is involved centromere throughout the cell cycle and transient proteins that in the packaging of centromeric chromatin and that the protein are present at various stages (1). Three of the best-studied may provide an early epigenetic marker for centromere forma- constitutive proteins are centromere proteins CENP-A, tion (29). CENP-B, and CENP-C. CENP-A is a 17-kDa histone H3-like Only limited functional data are available for CENP-A. Mi- croinjection of antibodies raised against the N terminus of protein involved in centromeric nucleosome formation (ref. 2; ͞ described below). CENP-B is an 80-kDa protein that binds a CENP-A into HeLa cells within 3 hr of G1 S release resulted in 17-bp motif known as the CENP-B box, which is present in interphase arrest (30). Highly condensed nuclei, granular cyto- human ␣-satellite and mouse minor satellite DNA (3, 4). Gene plasm, and loss of cell division capability were observed. Anti- knockout analysis of Cenpb in mice indicates that this protein is body injection in midinterphase did not disrupt mitosis; however, not essential (5–7), although a decrease in body weight and testis a mitotic lag was observed possibly because of the antibody size accompanied protein deficiency (5). CENP-C is a 140-kDa interfering with microtubule attachment (31). Studies on CSE4p protein that interacts with chromatin at the inner kinetochore (chromosome segregation protein), an S. cerevisiae homolog of plate (8). In vitro DNA binding studies suggest that CENP-C may CENP-A, have demonstrated the protein to be a component of bind to DNA (9). CENP-C null mutation results in embryonic the core centromere (32). Mutation in CSE4p results in misseg- lethality at 3.5 days postconception (pc), with a missegregation regation and cell arrest in mitosis; however, the increase in phenotype and metaphase arrest (10, 11). Metaphase arrest also chromosome loss is slight (33). The arrest phenotype is consis- is observed after microinjection of anti-CENP-C antibodies at tent with a specific cell division block that appears to occur after interphase (12). CENP-C shares a region of homology with Mif2, the mitotic spindle has formed but before the onset of anaphase a Saccharomyces cerevisiae protein. Mutations in the MIF2 gene (33). The arrested cells have a 2n DNA content, indicating that result in defective chromosome segregation and delayed pro- DNA replication has taken place before arrest. gression through mitosis (13). However, CENP-C alone is not sufficient to induce centromeric formation (14). This paper was submitted directly (Track II) to the PNAS office. A number of transient centromere proteins now have been Abbreviations: ES, embryonic stem; CENP, centromere protein; INCENP, inner CENP; pc, described (1, 15–19). Of particular relevance to the present study postconception; IRES, internal ribosome-entry site. involving the use of the gene targeting technique is the inner *To whom reprint requests should be addressed. E-mail: [email protected]. CENP (INCENP). This protein localizes to the centromere at The publication costs of this article were defrayed in part by page charge payment. This early mitosis and is present on the metaphase plate at the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. metaphase-anaphase transition (20). Gene disruption in mice §1734 solely to indicate this fact. 1148–1153 ͉ PNAS ͉ February 1, 2000 ͉ vol. 97 ͉ no. 3 Downloaded by guest on September 29, 2021 Fig. 2. Southern blotting and PCR genotyping of cell line, tail, and embryo DNA. (a) Southern blot analysis of putative targeted ES cell colonies after EcoRI digestion and probed with an external probe (see Fig. 1 b and d). (b) PCR analysis of mouse tail DNA showing a wild-type product of 455 bp detected by WT-1 and WT-2 primers, and a targeted product of 750 bp detected by N-1 and Fig. 1. Targeted disruption of the mouse Cenpa gene. (a) The mouse Cenpa WT-2 primers. SA, splice-acceptor site. (c) Nested PCR of mouse embryos protein showing the different subdomains, in particular those at the C termi- resulting in a 135-bp wild-type product when using primers MA1, MA2, MA3, nus that are required for centromere targeting. Our targeting construct (see and MA4, and a 248-bp targeted product when using primers GF1, GR1, GF2, below) was designed to delete amino acids 29–64 (gray box), which will and GR2. effectively remove the entire centromere-targeting domain. (b) A restriction map of the Cenpa gene. The exons are denoted by black boxes (23).(c) The GENETICS gene replacement construct, where the selectable marker cassette consists of by Southern blot (Fig. 2a). Chimeric mice were produced as a splice-acceptor site (SA), a picornaviral IRES, a lacZ-neomycin-resistance fusion gene, and a simian virus 40 polyadenylation sequence (PA). (d) The described (21). Mice were genotyped by PCR. DNA was ex- Cenpa locus after gene disruption. The positions of external probes used in tracted from mouse tails as described (21). Primers designed by Southern analysis are shown and the expected size fragments are 7.9-kb using GENEWORKS were: WT-1 (5Ј-TCAGACACTGCGCA- wild-type allele and a 4.8-kb targeted allele. ATG and TAA are translation start GAAGAC); WT-2 (5Ј-GAGCTTAGGAACTGGCATGG); and stop codons, respectively. Restriction enzymes used were SacI (S), SalI (Sa), and N-1 (5Ј-TTCTATCGCCTTCTTGACGAG) (Fig. 2b). EcoRI (E), XbaI (Xb), XhoI (Xh), KpnI (K), NheI (Nh), and SpeI (Sp). Genotyping of Preimplantation Embryos. Preimplantation embryos To further understand the role of CENP-A in centromere were obtained from heterozygous mice. Breeding pairs were function, we have used the technique of gene targeting by examined daily for vaginal plugs (an indicator of 0.5 days homologous recombination to enable the production of Cenpa gestation) and denoted as 0.5 days pc. A nested PCR protocol null mice. Our analysis of Cenpa null mutants has enabled us to was designed and used for the amplification of the 2.5-day elucidate the involvement of this protein in mitotic cell division embryonic DNA. Embryos were flushed and transferred to PCR ␮ and, in particular, its role in kinetochore assembly. tubes in 25 lofdH2O. Nest 1a: denaturation at 95°C for 15 min. ϫ Nest 1b: addition of 10 buffer (containing 15 mM MgCl2) Methods (Perkin–Elmer), 0.2 mM dNTP, 250 ng of wild-type primers Construction of Targeting Vectors.